The mechanisms involved in Ehrlichia chaffeensis internalization, establishment of intracellular infection, persistence, and subversion of antimicrobial defenses are not well understood, but appear to be dependent on secreted ehrlichial effector proteins. E. chaffeensis utilizes T1S to export tandem repeat protein (TRP) effectors that escape the ehrlichial vacuole through an unknown mechanism. TRP47 and TRP120 are highly antigenic proteins that we recently determined interact with a diverse array of host proteins including, but not limited to, post translational modification ligases and epigenetic regulators. PCGF5, a component of the polycomb-repressive complex (PRC) involved in epigenetic regulation of Hox genes, strongly interacts with these TRP effectors. We have determined that polycomb group (PcG) proteins are recruited to ehrlichial vacuole and are differentially degraded. Moreover, consistent with PcG protein degradation, the PRC1- mediated H2AK119ub repressive histone mark is significantly decreased, while PRC2-mediated mark H3K27me3 is downregulated in a gene-specific manner. The objective of this proposal is to demonstrate E. chaffeensis modulates PRC controlled Hox genes through TRP-mediated degradation of PcG proteins, thereby modulating associated repressive epigenetic histone modifications. Through this investigation we will establish the mechanism by which E. chaffeensis exploits an evolutionarily conserved group of epigenetic regulators to establish a cell type-specific gene expression program that promotes intracellular survival. In the first aim, we will examine changes in PcG proteins during E. chaffeensis infection and associated epigenetic histone marks, determine molecular TRP interactions involved in PcG recruitment to the vacuole, and characterize components of PcG-protein complexes in normal and infected contexts to determine TRP induced changes. In the second aim, expression of PcG regulated Hox genes will be examined during E. chaffeensis infection by microarray, the role of specific PcG isoforms in regulating Hox gene expression during infection and survival will be examined using iRNA, and epigenetic marks will be investigated on target genes to determine the role of TRP-PcG mediated regulation. The exploitation of PcG transcriptional regulation in mononuclear phagocytes by a bacterial pathogen is novel, for which E. chaffeensis represents a model system to investigate this complex pathogen-host interaction. This investigation will define the targets and mechanisms of TRP-mediated transcriptional reprogramming strategies, and determine the roles of PcG and epigenetic marks in modulating host gene expression in order to create a cell type-specific prosurvival program. Characterization of the host cell pathways modulated by ehrlichial effectors and the molecular mechanisms involved will expand our understanding of the general principles that underlie the cell biology of infection and surviva strategies utilized by intracellular microbes. This knowledge is necessary to identify novel host targets for therapeutics based on mechanistically defined host-pathogen interactions potentially utilized by a wide range of microbes.